This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding carbohydrate biosynthetic enzymes in plants and seeds.
Brittle-1 is one of several corn genes that, when mutated, cause the accumulation of sugars, rather than starch, in developing corn seeds. It has been shown that the brittle-1 gene encodes a plastidic membrane transporter that is involved in the transport of ADP-glucose from the cytosol to the plastid where it is used for starch biosynthesis (Shannon et al. (1998) Plant Physiol 117:1235-1252). In corn, the mutant phenotype suggests that inactivation of the brittle-1 gene causes a reduction in starch accumulation. This reduction in starch accumulation presumably causes an increase in concentration of various sugars which in turn provides a large available pool of carbon for other metabolic pathways (Sullivan, T. D. et al. (1991) Plant Cell 3(12):1337-1348; Sullivan, T. D. et al. (1995) Planta 196(3):477-484). By manipulating the level of the brittle-1 protein in cells, it may be possible to modulate the level (higher or lower) of starch accumulation in seeds, thereby controlling the level of undesirable carbohydrate concentration.
Callose or 1,3-beta-D-glucan synthesis is stimulated in response to infection by a plant pathogen. Callose appears to act as a physical barrier against plant pathogens (Beffa, R. S. et al. (1996) Plant Cell 8(6):1001-1011). It has been recently shown that plant mutants that do not produce callose efficiently or mutants that degrade callose more rapidly than normal have increased risk of infection by specific pathogens (Beffa, R. S. et al. (1996) Plant Cell 8(6):1001-1011). These observations suggest that by modulating the level of callose in a plant cell it may be possible to manipulate plant host defense systems. For example, it may be possible to cause an increase in the production of callose and thus provide enhanced disease resistance. Furthermore, a high beta-glucan content in plant material is desirable because beta-glucan is a major component of soluble fiber. Soluble fiber is important in a healthy diet because in general soluble fiber has been shown to reduce cholesterol levels in humans (Fastnaught, C. E. et al. (1996) Crop Science 36:941-946).
Few of the genes encoding these enzymes in barley, corn, soybeans, rice and wheat have been isolated and sequenced. For example, no barley, soybean or wheat genes have been reported for the brittle-1 protein and no corn, rice, Vernonia or wheat genes have been reported for 1,3-beta-D-glucan synthase. Accordingly, the availability of nucleic acid sequences encoding all or a portion of these enzymes would facilitate studies to better understand carbohydrate metabolism and function in plants, provide genetic tools for the manipulation of these biosynthetic pathways, and provide a means to control starch and 1,3-beta-D-glucan synthesis in plant cells.
The present invention concerns an isolated polynucleotide comprising: (a) a first nucleotide sequence encoding a first polypeptide comprising at least 200 amino acids, wherein the amino acid sequence of the first polypeptide and the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:10 have at least 70%, 80%, 85%, 90%, or 95% identity based on the Clustal alignment method, (b) a second nucleotide sequence encoding a second polypeptide comprising at least 200 amino acids, wherein the amino acid sequence of the second polypeptide and the amino acid sequence of SEQ ID NO:14 have at least 85%, 90%, or 95% identity based on the Clustal alignment method, (c) a third nucleotide sequence encoding a third polypeptide comprising at least 300 amino acids, wherein the amino acid sequence of the third polypeptide and the amino acid sequence of SEQ ID NO:18 have at least 70%, 80%, 85%, 90%, or 95% identity based on the Clustal alignment method, or (d) the complement of the first, second, or third nucleotide sequence, wherein the complement and the first, second, or third nucleotide sequence contain the same number of nucleotides and are 100% complementary. The first polypeptide preferably comprises the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:10, the second polypeptide preferably comprises the amino acid sequence of SEQ ID NO:14, and the third polypeptide preferably comprises the amino acid sequence of SEQ ID NO:18. The first nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:9, the second nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:13, and the third nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:17. The first, second, and third polypeptides preferably are brittle-1 homologs.
In a second embodiment, the present invention relates to a chimeric gene comprising any of the isolated polynucleotides of the present invention operably linked to a regulatory sequence, and a cell, a plant, and a seed comprising the chimeric gene.
In a third embodiment, the present invention relates to a vector comprising any of the isolated polynucleotides of the present invention.
In a fourth embodiment, the present invention relates to an isolated polynucleotide fragment comprising a nucleotide sequence comprised by any of the polynucleotides of the present invention, wherein the nucleotide sequence contains at least 30, 40, or 60 nucleotides.
In a fifth embodiment, the present invention concerns an isolated polypeptide comprising: (a) a first amino acid sequence comprising at least 200 amino acids, wherein the first amino acid sequence and the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:10 have at least 70%, 80%, 85%, 90%, or 95% identity based on the Clustal alignment method, (b) a second amino acid sequence comprising at least 200 amino acids, wherein the second amino acid sequence and the amino acid sequence of SEQ ID NO:14 have at least 85%, 90%, or 95% identity based on the Clustal alignment method, or (c) a third amino acid sequence comprising at least 300 amino acids, wherein the third amino acid sequence and the amino acid sequence of SEQ ID NO:18 have at least 70%, 80%, 85%, 90%, or 95% identity based on the Clustal alignment method. The first amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:10, the second amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:14, and the third amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:18. The polypeptide preferably is a brittle-1 homolog.
In a sixth embodiment, the present invention relates to a method for transforming a cell comprising transforming a cell with any of the isolated polynucleotides of the present invention, and the cell transformed by this method. Advantageously, the cell is eukaryotic, e.g., a yeast or plant cell, or prokaryotic, e.g., a bacterium.
In a seventh embodiment, the present invention relates to a method for producing a transgenic plant comprising transforming a plant cell with any of the isolated polynucleotides of the present invention and regenerating a plant from the transformed plant cell, the transgenic plant produced by this method, and the seed obtained from this transgenic plant.
In an eighth embodiment, the present invention relates to a virus, preferably a baculovirus, comprising any of the isolated polynucleotides of the present invention or any of the chimeric genes of the present invention.
In a ninth embodiment, the invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a brittle-i homolog protein or enzyme activity in a host cell, preferably a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; (b) introducing the isolated polynucleotide or the isolated chimeric gene into a host cell;(c) measuring the level of the brittle-1 homolog protein or enzyme activity in the host cell containing the isolated polynucleotide; and (d) comparing the level of the brittle-1 homolog protein or enzyme activity in the host cell containing the isolated polynucleotide with the level of the brittle-1 homolog protein or enzyme activity in the host cell that does not contain the isolated polynucleotide.
In a tenth embodiment, the invention concerns a method of obtaining a nucleic acid fragment encoding a substantial portion of a brittle-1 homolog protein, preferably a plant brittle-1 homolog protein, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:3, 9, 13, and 17, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a substantial portion of a brittle-1 homolog protein amino acid sequence.
In an eleventh embodiment, this invention relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a brittle-1 homolog protein comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
In a twelfth embodiment, this invention concerns a method for positive selection of a transformed cell comprising: (a) transforming a host cell with the chimeric gene of the present invention or an expression cassette of the present invention; and (b) growing the transformed host cell, preferably a plant cell, such as a monocot or a dicot, under conditions which allow expression of the brittle-1 homolog protein polynucleotide in an amount sufficient to complement a null mutant to provide a positive selection means.
In a thirteenth embodiment, this invention relates to a method of altering the level of expression of a brittle-1 homolog protein in a host cell comprising: (a) transforming a host cell with a chimeric gene of the present invention; and (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of the brittle-1 homolog protein in the transformed host cell.